Advances in stem cell technology, such as the isolation and use of human embryonic stem (hES) cells, have become an important new subject of medical research. hES cells have a demonstrated potential to differentiate into any and all of the cell types in the human body, including complex tissues. This ability of hES cells has led to the suggestion that many diseases resulting from the dysfunction of cells may be amenable to treatment by the administration of hES-derived cells of various differentiated types (Thomson et al., Science 282:1145-7, (1998)). Nuclear transfer studies have demonstrated that it is possible to transform a somatic differentiated cell back to a totipotent state such as that of ES or ED cells (Cibelli et al., Nature Biotech 16:642-646, (1998)). The development of technologies to reprogram somatic cells back to a totipotent ES cell state such as by nuclear transfer offers a means to deliver ES-derived somatic cells with a nuclear genotype of the patient (Lanza et al., Nature Medicine 5:975-977, (1999)). It is expected that such cells and tissues would not be rejected, despite the presence of allogeneic mitochondria (Lanza et al., Nature Biotech 20:689-696, (2002)). Nuclear transfer also allows the rebuilding of telomere repeat length in cells through the reactivation of the telomerase catalytic component in the early embryo (Lanza et al., Science 288:665-669, (2000)). Nevertheless, there remains a need for improvements in methods to reprogram animal cells that increase the frequency of successful and complete reprogramming and reduce the dependence on the availability of human oocytes.
Because of the relative difficulty of obtaining large numbers of human oocytes, there has been considerable interest in determining whether other germ-line cells, such as cultured ES cells, or cytoplasm from said cells, could be used to reprogram somatic cells. Such cells would have an important advantage over oocytes as a means of inducing reprogramming in that they can be easily expanded in number in vitro. The restoration of expression of at least some measured embryonic-specific genes has been observed in somatic cells following fusion with ES cells (Do and Scholer, Stem Cells 22:941-949, (2004); Do and Scholer, Reprod. Fertil. Dev. 17:143-149, (2005)). However, the resulting cells are hybrids, often with a tetraploid genotype, and therefore not suited as normal or histocompatible cells for transplant purposes. Indeed, one of the proposed purposes of generating autologous totipotent cells is to prevent the rejection of ES-derived cells. Using the techniques described in these published studies, the ES cells used to reprogram a patient's cell would therefore likely add alleles that could generate an immune response leading to rejection. Nevertheless, the evidence that ES cells can reprogram somatic cell chromosomes has excited researchers and generated a new field of research called “fusion biology” (Dennis, Nature 426:490-491, (2003)).
Another potential source of cells capable of reprogramming human somatic cells with a greater ease of availability than human oocytes are oocytes of animal species. The demonstration of the restoration of totipotency in somatic cells by nuclear transfer across species (Lanza et al., Cloning 2:79-90, (2000)) opens the possibility of identifying animal oocytes that can be easily obtained for use in reprogramming human cells (Byrne et al., Curr Biol 13:1206-1213, (2003)). However, likely because of molecular differences between the species, cross species nuclear transfer, although possible, is often even more inefficient than same-species nuclear transfer.
Among the many molecular alterations that occur following somatic cell nuclear transfer, some of the more critical alterations are the reprogramming of the chromatin through poorly-understood mechanisms in the recipient oocyte and remodeling of the proteins of the nuclear envelope. The nuclear envelope includes the inner nuclear membrane (INM) and outer nuclear membrane (ONM), nuclear pore complexes (NPCs), and nuclear lamina. The proteins of the nuclear envelope, in particular those proteins of the lamina, differ between somatic and germ-line cells and play an important role in regulating the cell cycle, monitoring DNA damage checkpoint pathways, and regulating cell differentiation. In particular, the protein subunits of the lamina include the type V intermediate filament proteins, lamin A/C and B, which form a meshwork internal to the INM (Foisner, J. Cell Sci. 114:3791-3792, (2001)). Some of these proteins, such as lamin A/C, play an important role in regulating chromosomal integrity, DNA damage checkpoints, and telomere status signaling through their interactions with the WRN helicase, POT1, Tel1, and Tel2. In germ-line cells that are telomerase positive, or where telomerase is utilized, the nuclear matrix lacks lamin A/C or otherwise allows tandemly-repeated sequences of DNA to be repaired and, in the case of the telomere, to be lengthened by telomerase. Other proteins associated with the INM include the family of lamina associated polypeptides (LAPs) including lamina-associated protein 1 (LAP1, of which there are at least three isoforms (α, β, and γ)), LAP2 (with at least six isoforms) and emerin (which when mutated leads to abnormal muscle differentiation and Emery-Dreifuss muscular dystrophy). Other proteins associated with the INM include the ring finger binding protein (RFBP), otefin, germ cell-less (GCL) and nurim. The lamins are known to play an important role in regulating the function of transcriptional regulators such as the retinoblastoma protein (pRB) and LBR which in turn can bond heterochromatin protein 1 (HP1). By way of example of the need to remodel the nuclear envelope in order to reprogram a differentiated somatic cell to an undifferentiated state, undifferentiated germ-line cells generally lack the presence of lamin A, while germ-line cells contain proteins such as germ cell-less (GCL) and lamin C2, which are often not expressed in differentiated somatic cells (Furukawa et al., Exp. Cell Res. 212:426-430, 1994). Incomplete remodeling of the nuclear envelope would contribute to the inefficiency or incomplete reprogramming of cells using existing technologies.
Therefore, each of the technologies to reprogram human somatic cells known in the art have their own unique difficulties. SCNT provides a satisfactory level of reprogramming but is limited by the number of human oocytes available to researchers. Cross-species nuclear transfer and cell fusion technologies are not generally limited in the cells used in reprogramming but are limited by the degree of successful reprogramming or the robustness of the growth of the resulting reprogrammed cells. Therefore, there remains a need for improved technologies to both increase the frequency and quality of reprogramming of differentiated somatic cells and of producing reprogrammed cells that are capable of expansion in vitro in order to obtain a useful number of cells for research, testing for quality control, and for use in cell therapy. The present invention combines aspects of several existing technologies already known in the art in a novel and non-obvious manner to provide a means of reprogramming differentiated cells as effectively or more effectively than SCNT and to provide a more acceptable and cost-effective substitute for oocytes as the vehicle for reprogramming. The present invention achieves these goals in part by using cells that are easily and inexpensively obtained in unlimited quantities and a technology that can be scaled such that thousands or millions of fusions can be performed simultaneously, thereby increasingly the probability of a successful final outcome. Additionally, the present invention provides a technique that facilitates the reactivation of telomerase and an extension of telomere length, thereby restoring cell replicative lifespan. The present invention further provides an assay that allows for the analysis of what components in undifferentiated and germ-line cells are critical for nuclear reprogramming. The invention also provides a procedure that can be automated through robotics to reduce cost and improve quality control.